Abstract The Tie family of receptor tyrosine kinases (RTKs) are involved in both vascular homeostasis and in angiogenesis. Both the receptors and their angiopoietin (Ang) ligands are attractive targets for pharmacologic intervention in cancer, inflammation and other disease states. An impediment to development of therapeutic agents is the current incomplete understanding of the mechanisms of activation of Tie receptors. Whereas RTKs such as EGFR, Kit and FGFR are well known to be regulated by growth factor-induced dimerization, for the Tie receptors, studies to date have failed to reveal the mechanism of ligand-induced receptor activation. The activating, oligomeric Ang ligands all bind to Tie2, with signaling outcomes that are context dependent and are modulated by co-receptors including the orphan family member Tie1. We have shown that the extracellular region (ECR) of Tie2 forms a ligand-independent dimer that is mediated by its membrane-proximal fibronectin type III (FNIII) domains, and is essential for Tie2 activation in cells. The oligomeric Ang ligands are known to bind to the membrane distal domains of Tie2. The Tie receptors thus differ from most RTKs in that an oligomeric ligand regulates an already oligomeric receptor. Whether signaling arises through allosteric changes in a receptor dimer or the promotion of receptor crosslinking or clustering remains unclear (but is a focus of our proposal). For several RTKs with immunoglobulin domains in their membrane-proximal regions (KIT, PDGFR and Fms/CSF1-R), homotypic interactions between membrane-proximal regions are important for receptor dimerization and activation, and disruption of these interactions by antibody binding or mutation blocks receptor activation. We propose that interactions involving the membrane-proximal FNIII domains of Tie receptors can also be targeted therapeutically. Further, we suggest that Tie receptors may serve as a prototype for the 40% of human RTKs that have membrane-proximal FNIII domains. To gain insight into how the oligomeric Ang ligands bind and activated a dimeric RTK, we will use a combination of structural, biochemical and cellular approaches to address the consequences of binding of defined, oligomeric Ang ligands to the Tie2 ECR dimer. We will also determine how ligand binding and signaling are influenced by the presence of Tie1 and the RPTP?, both of which interact directly with Tie2. Our overall goal is to build a detailed molecular understanding of how the interactions mediated by the ECR of Tie2 regulate receptor activity, and to exploit this information to develop antibody modulators of Tie receptor activation. Our specific aims ask three questions: 1. How do angiopoietin ligands regulate Tie2? 2. How do Tie1 and RPTP? modulate the effects of Ang ligands on Tie2 activity? 3. Can specific antibodies selectively block subsets of Tie2 interactions?